Host Cells Used For Production of Recombinant Protein

ABSTRACT

It is an object of the present invention to provide host cells, which are able to proliferate in a serum free medium, which are able to synthesize and secrete a large amount of protein of interest, and which are able to produce a glycoprotein having a sugar chain structure. The present invention provides an established cell line, which is established from distal tubular epithelial cells derived from a mammal.

TECHNICAL FIELD

The present invention relates to host cells used for production of arecombinant protein, and a use of the aforementioned host cells. Thepresent invention particularly relates to host cells used for productionof a recombinant protein established from a mammal, and a use of theaforementioned host cells.

BACKGROUND ART

The functional analysis of a gene whose functions are unknown, it isessential to obtain a recombinant protein encoded by the aforementionedgene. The currently used expression systems for production ofrecombinant proteins include a prokaryotic system, a eukaryotic system,and a cell-free system. Such prokaryotic systems used as hosts include:Escherichia coli and related bacteria (e.g. E. coli, bacteria of genusPseudomonas, bacteria of genus Zymomonas, bacteria of genus Thermus,etc.); and Bacillus subtilis and related bacteria) (e.g. Actinomycetes,lactic acid bacteria, amino acid-producing bacteria, acetic acidbacteria, thermophilic bacteria, etc.). Such eukaryotic systems used ashosts include yeast, filamentous bacteria, cultured plant cells,cultured insect cells (baculovirus vector), and cultured animal cells.

Among the aforementioned expression systems, mass production systems ofrecombinant proteins using microorganisms such as Escherichia coli oryeast have been established, and several proteins have been used asmedicaments. However, in eukaryotes, and in particular, in the case ofhuman proteins, it has been known that addition of characteristic sugarchains and a complicated processing conducted after the synthesis ofsuch human proteins are essential for activity of the proteins. Thus,there may be cases where it is difficult to produce functional proteinsfrom microorganisms.

Moreover, even in a case where a protein does not undergoposttranslational modification, when a human gene is expressed in anEscherichia coli expression system, inclusion bodies may be often formeddue to differences in codon usage or conformation of proteins. Thus,even if a gene can be obtained, an enormous time is required forobtaining the gene product (protein). This causes a bottleneck in manycases.

On the other hand, when a protein of interest is produced using insectcells or cultured animal cells, this is problematic in terms of (1) alow amount of production, (2) a limited range of genes to be expressed,(3) high costs, and the like. Accordingly, time and effort are requiredfor obtaining a protein to be subjected to screening.

Furthermore, when a large-scale production of proteins is carried outusing animal cells, this method has been problematic in the followingrespects. (1) In the case of transient expression of a protein, theprotein of interest can be obtained in a short time. However, the amountof a protein that can be produced by a single transfection is limited,and a scale-up is not easy. Thus, large quantities of cells andtransfection reagents are required. (2) Cells that highly expressproteins over a long period of time can be obtained by producing a cellline where the introduced gene is stably expressed. However, this methodhas been problematic in that it takes a long time (several months) toproduce a cell line into which a foreign gene has been stablyintroduced.

A glycoprotein with a molecular weight of 60 kDa named as “Cauxin” isegested in the urine of cats at a high concentration of approximately 1mg/ml. It has been reported that Cauxin is generated in distal tubularepithelial cells and is then secreted from an apical membrane(Non-Patent Document 1).

Non-Patent Document 1: Miyazaki, M., Kamiie, K., Soeta, S., Taira, H.,and Yamashita, T.: Molecular cloning and characterization of a novelcarboxylesterase-like protein that is physiologically present at highconcentration in the urine of domestic cats (Felis catus) Biochem. J.,370, 101-110 (2003).

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

It is an object of the present invention to provide host cells, whichare able to proliferate in a serum free medium, which are able tosynthesize and secrete a large amount of protein of interest, and whichare able to produce a glycoprotein having a sugar chain structure.

Means for Solving the Problems

As a result of intensive studies directed towards achieving theaforementioned object, the present inventors have found that anestablished cell line, which is established from cat-derived distaltubular epithelial cells, is able to proliferate in a serum free medium,is able to synthesize and secrete a large amount of protein of interest,and is also able to produce a glycoprotein having a sugar chainstructure. The present invention has been completed based on suchfindings.

Thus, the present invention provides an established cell line, which isestablished from distal tubular epithelial cells derived from a mammal.

Preferably, the established cell line of the present invention is usedas a host cell for production of a recombinant protein.

Preferably, the established cell line of the present invention isderived from a cat.

Preferably, the established cell line of the present invention has agene encoding a foreign immortality-imparting factor.

Preferably, the established cell line of the present invention has aforeign selective marker gene.

Preferably, the established cell line of the present invention is ableto proliferate in a serum free medium.

Preferably, the established cell line of the present invention is a cellhaving an accession No. FERM BP-10643.

According to another aspect, the present invention provides atransformant, which is obtained by introducing a gene encoding a proteinof interest to be expressed into the established cell line of thepresent invention as mentioned above.

According to further another aspect, the present invention provides amethod for producing a recombinant protein using the established cellline of the present invention as mentioned above.

Preferably, a foreign gene is introduced into the established cell lineof the present invention as mentioned above to produce transformedcells, the obtained transformed cells is cultured so as to allow thecells to produce a recombinant protein encoded by the foreign gene, andthe produced recombinant protein can be recovered.

Preferably, the produced recombinant protein is modified with a sugarchain.

According to further another aspect, the present invention provides arecombinant protein, which is produced by the method of the presentinvention as mentioned above.

BEST MODE FOR CARRYING OUT THE INVENTION

The embodiments of the present invention will be described in detailbelow.

The cells of the present invention are established cells which areestablished from mammal-derived distal (renal) tubular epithelial cells.Examples of such a mammal include cat, dog, rabbit, guinea pig, hamster,rat, mouse, and human. Of these, cat is preferable.

The cells of the present invention can be collected from cat kidney, forexample. The kidney excised from a cat is fragmented in a Joklik buffer(111 mM NaCl, 24 mM NaHCO₃, 10 mM Na₂HPO₄, 5.4 mM KCl, 1 mM Mg₂SO₄, and11 mM glucose), and the fragmented tissue section is treated with acollagenase digestive fluid. Thereafter, cell masses are removed fromthe thus fragmented cells. The cells are then recovered bycentrifugation. Subsequently, the cells are suspended in Eagle's MEMmedium 1 (Nissui), and distal tubular cells are then separated byPercoll density gradient centrifugation, so that the aforementionedcells can be recovered. The cells are washed with Eagle's MEM medium 1so as to remove Percoll, and the resultant cells are then suspended in aDME-F12 medium, followed by culture on a collagen-coated dish.

An immortality-imparting factor and a selective marker gene areintroduced into the thus isolated distal tubule-derived cells, so as toestablish a cell line. The term “immortality-imparting factor” is usedto mean a factor that is introduced into cells to immortalize them.Examples of such an immortality-imparting factor used herein include anEpstein-Barr virus (EBV) gene, a Simian virus 40 (SV40) T antigen gene,an SV40 large T antigen gene, an adenovirus E1A gene, an adenovirus E1Bgene, a human papillomavirus (HPV) E6 gene, an HPV E7 gene, and atelomerase reverse transcriptase protein TERT) gene. As a selectivemarker gene, a drug resistance gene, an auxotrophic gene, and the likecan be used. Examples of such genes used herein include a neomycinresistance gene, a puromycin resistance gene, a hygromycin resistancegene, and a zeocin resistance gene. A gene encoding animmortality-imparting factor and a selective marker gene can beintroduced by a common transfection method. After completion of thetransfection, the used medium is exchanged with a selective medium (inthe case of introducing a drug resistance gene, a medium containing theaforementioned drug is used, for example), and a cell line, into which aselective marker gene has been stably introduced and which is able tostably proliferate, is selected, thereby establishing the cell line ofthe present invention.

As an example of the cells of the present invention, FKD cells(identification name: FKD) obtained in the example as given below weredeposited with the National Institute of Advanced Industrial Science andTechnology, an Independent Administrative Institution under the Ministryof Economy, Trade and Industry (at the AIST Tsukuba Central 6, Higashi1-1-1, Tsukuba, Ibaraki, Japan, (Post code: 305-8566)) under accessionNo. FERM P-20605 on Jul. 27, 2005. Thereafter, this deposition wastransferred to an international deposition under “Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure” on Jul. 19, 2006 under an accession No.FERM BP-10643.

The thus established cell line of the present invention can be culturedby a common method for culturing animal cells. As a medium used in theculture, commonly used RPM11640 medium [The Journal of the AmericanMedical Association, 199, 519 (1967)], Eagle's MEM medium [Science, 122,501 (1952)], DMEM medium [Virology, 8, 396 (1959)], DMEM-F12 medium, andthe like can be used. Otherwise, a medium obtained by adding fetalbovine serum or the like to such a medium can also be used. However,since the cells of the present invention are able to proliferate even ina serum free medium, it is not necessary to add serum. It may also bepossible to add insulin, transferrin, serine, or the like to such amedium, as appropriate.

In general, culturing can be carried out under conditions such as pH 6to 8, a temperature between 30° C. and 40° C., and the presence of 5%CO₂. In addition, during such culturing, antibiotics such as neomycinmay be added to a medium, as necessary. Culturing may also be carriedout on a collagen-coated dish.

In order to allow a recombinant protein of interest to express in thecells of the present invention, first, a gene encoding such arecombinant protein is inserted into a site downstream of a promoter ina suitable expression vector. Thereafter, the expression vector, intowhich the aforementioned gene has been inserted, is introduced into thecells of the present invention.

As an expression vector, a vector, which is capable of autonomouslyreplicating in the cells of the present invention or is capable of beingincorporated into the chromosome and which comprises a promoter at asite for transcribing the aforementioned gene of interest, can be used.

Examples of such an expression vector include pCH110 (PL), pcDNAI, pcDM8(Funakoshi Corp.), pcDNAI/AmP (Invitrogen), pREP4 (Invitrogen), pCDNA3Invitrogen), pCMVscript (Stratagene), pSG5 (Stratagene), pZeoSV2(Invitrogen), pEF1/V5 HisA/B/C Invitrogen), pEF-Bos-Myc, pREP4(Invitrogen), and pBK-RSV (Stratagene).

Any type of promoter can be used, as long as it can be expressed inanimal cells. Examples of such a promoter include a cytomegalovirus(human CMV) IE (immediate early) gene promoter, an SV40 early promoter,a retrovirus promoter, a metallothionein promoter, a heat shockpromoter, and an SRα promoter. Moreover, a human CMV IE gene enhancermay also be used together with such a promoter.

As a method for introducing a recombinant expression vector into thecells of the present invention, a common method for introducing DNA intoanimal cells can be used. Examples of such a method include anelectroporation method, a calcium phosphate method, and a lipofectionmethod.

In addition, as a method for expressing a recombinant protein, directexpression as well as secretory production, expression of a fusionprotein and the like can be carried out.

When a recombinant protein is allowed to express in the cells of thepresent invention, a protein, to which sugar or a sugar chain is added,can be obtained.

A transformant obtained by introducing a gene encoding a protein ofinterest to be expressed into the cells of the present invention iscultured in a preferred medium, and a protein of interest is producedand accumulated in the culture. Thereafter, the protein is collectedfrom the culture, so as to produce the protein of interest. The thusproduced protein is also included in the scope of the present invention.

As a method for culturing the transformant of the present invention in amedium, a common method used in the culture of a host can be applied.

As a medium for culturing a transformant, commonly used RPM11640 medium[The Journal of the American Medical Association, 199, 519 (1967)],Eagle's MEM medium [Science, 122, 501 (1952)], DMEM medium [Virology, 8,396 (1959)], DMEM-F12 medium, and the like can be used. Otherwise, amedium obtained by adding fetal bovine serum or the like to such amedium can also be used. However, since the cells of the presentinvention are able to proliferate even in a serum free medium, it is notnecessary to add serum.

In general, culturing can be carried out for 1 to 7 days underconditions such as pH 6 to 8, a temperature between 30° C. and 40° C.,and the presence of 5% CO₂. In addition, during such culturing,antibiotics such as neomycin may be added to a medium, as necessary.

In order to isolate and purify a recombinant protein of interest fromthe culture of the transformant of the present invention, a commonmethod for isolating and purifying a protein may be used.

When a recombinant protein is expressed in cells in a soluble state, forexample, the cells are recovered by centrifugation after completion ofthe culturing, and they are then suspended in a water-based buffer.Thereafter, the cells are crushed using an ultrusonic disintegrator, aFrench press, a Manton Gaulin homogenizer, a Dyno mill, etc., so as toobtain a cell-free extract. The cell-free extract is centrifuged toobtain a supernatant. Thereafter, the obtained supernatant is subjectedto common methods for isolating and purifying a protein; namely, asolvent extraction method, a salting-out method using ammonium sulfate,a desalination method, a precipitation method using an organic solvent,anion exchange chromatography using a resin such as diethylaminoethyl(DEAE) sepharose or DIAION HPA-75 (manufactured by Mitsubishi KaseiCorp.), cation exchange chromatography using a resin such as S-SepharoseFF (manufactured by Pharmacia), hydrophobic chromatography using a resinsuch as butyl sepharose or phenyl sepharose, a gel filtration methodusing a molecular sieve, affinity chromatography, a chromatofocusingmethod, electrophoresis such as isoelectric electrophoresis, etc. Suchmethods are used singly or in combination, so as to obtain a purifiedsample.

When a recombinant protein is expressed in cells in the form of aninsoluble form, the cells are recovered and are then crushed, followedby centrifugation, so as to obtain a precipitated fraction. Thereafter,a protein is recovered from the precipitated fraction by an ordinarymethod, and the insoluble form of the protein is solubilzed with aprotein denaturant. The solubilized solution is diluted or dialyzed tobe a solution that contains no protein denaturants or a solution thatcontains a protein denaturant whose concentration is too low to denaturethe protein, so that the protein is allowed to have a normalthree-dimensional structure. Thereafter, the same isolation andpurification methods as described above are applied, so as to obtain apurified sample.

When a recombinant protein or a derivative thereof such as asugar-modified derivative thereof is secreted outside the cells, theprotein or a derivative thereof such as a sugar chain-added derivativethereof can be recovered in the culture supernatant. That is to say, theculture is treated by the aforementioned methods such as centrifugationto obtain a soluble fraction. Thereafter, a purified sample can beobtained from the soluble fraction by the same above isolation andpurification methods.

The present invention will be described more in detail in the followingexamples. However, these examples are not intended to limit the scope ofthe present invention.

EXAMPLES Example 1 Isolation of Cat Renal Tubular Epithelial Cells

Cat distal tubule-derived epithelial cells were isolated as followsaccording to the method described in Soshiki Baiyo no Gijyutsu (2^(nd)edit) Nihon Soshiki Baiyo Gakkai Hen, Asakura Shoten, 7-4 Jinsaibo BaiyoHo (pp. 141-145) (issue Culture Techniques (2^(nd) edit) edited by theJapanese Tissue Culture Association, Asakura Shoten, 7-4 Renal CellCulture Method (pp. 141-145)). A cat was subjected to anesthesia, andthe kidney was aseptically excised. The excised kidney was fragmentedwith a knife in a Joklik buffer (111 mM NaCl, 24 mM NaHCO₃, 10 mMNa₂HPO₄, 5.4 mM KCl, 1 mM Mg₂SO₄, and 11 mM glucose). 30 ml of a 0.02%collagenase digestive fluid was added to 1 g of the fragmented tissuesection in a 100-ml Erlenmeyer flask, and the obtained mixture was thenstirred with a stirrer for 30 minutes. Thereafter, a supernatant wasdiscarded, and a fresh digestive fluid was added, followed by a furtherreaction for 30 minutes. The fragmented cells were filtrated throughgauze to remove cell masses, and the cells were then recovered bycentrifugation at 1,000 rpm for 5 minutes. The cells were suspended inEagle's MEM medium 1 (Nissui), and distal tubular epithelial cells wereseparated and recovered by Percoll density gradient centrifugation. Thecells were washed with Eagle's MEM medium 1 so as to remove Percoll, andthe resultant cells were then suspended in a DME-F12 medium, followed byculturing on a collagen-coated dish.

Example 2 Establishment of FKD Cells

Using Lipofectamine-Plus (Invitrogen), pSV3-neo (ATCC37150) wastransfected to the distal tubule-derived cells isolated in Example 1.Forty-eight hours after the transfection, the medium was exchanged witha G418-containing medium, and a stable mutation introduced cell line wasthen selected. The obtained FKD cells are shown in FIG. 1. Theproliferated cells were subjected to a passage, and the resultant cellswere then preserved in liquid nitrogen.

Example 3 Subculture (Passage) of FKD Cells

FKD cells were subcultured in a DMEM-F12 (+supplement) medium on acollagen-coated dish at 37° C. in the presence of 5% CO₂. When the cellsbecame 80% to 90% confluent, the medium was removed. The cells werewashed with PBS/EDTA, and 1 ml of PBS/EDTA was then added thereto,followed by incubation at 37° C. in the presence of 5% CO₂ for 5minutes. Subsequently, the cells were washed with 0.1% trypsin-PBS/EDTA,followed by incubation under the same above conditions. Thereafter, theresultant cells were suspended in a DMEM-F12 (+supplement) medium, andthe suspension was diluted and then cultured in a fresh collagen-coateddish. As a supplement (GIBCO ITS-X supplement), a product comprisinginsulin, transferrin and serine was used.

Example 4 Transfection Experiment Gene Transduction Efficiency; FIG. 2

In order to study transfection efficiency, an expression vector pCH110(Pharmacia) produced by binding LacZ to an SV40 promoter was used.Transfection was carried out using Lipofectamine-Plus (Invitrogen). Ascells, FKD cells, COS cells, and HeLa cells were used. Twenty-four hoursafter the transfection, the medium was removed, and the cells were thenrinsed with 1.5 ml of PBS once. Thereafter, 1.5 ml of a fixativesolution was added thereto, and the mixture was then left at rest for 10minutes, so as to fix the cells. Thereafter, the cells were rinsed with1.5 ml of PBS twice, and 1.25 ml of a staining solution (i-gal StainingKit (Invitrogen)) was then added thereto, followed by incubation. Threehours later, expression efficiency was confirmed from the ratio of thestained cells. The results are shown in FIG. 2. The FKD cells of thepresent invention exhibited a gene transduction efficiency higher thanthose of COS cells and HeLa cells.

Example 5 Production of Growth Curves of FKD Cells and COS Cells (FIG.3)

The growth of FKD cells in a serum free medium and in a serum medium andthe growth of COS cells in a serum medium were analyzed. Confluent FKDcells (6-cm collagen-coated dish (4.0×10⁶ cell)) were dispersed on thesame above dish, resulting in a cell number of 1:10 (4.0×10⁵ cells).Likewise, the same cells were dispersed in a serum medium on a 3.5-cmdish, resulting in a cell number of 1:10 (1.6×10⁵ cells). The cells weretreated with PBS/EDTA and 0.1% trypsin-PBS/EDTA, and they were thensuspended in 2 ml of a serum free medium (the cells in a serum mediumwere suspended in 1 ml of a serum medium). The cells were recovered andwere then centrifuged at 700 rpm for 10 minutes. Thereafter, asupernatant was discarded, and PBS and a trypan blue solution were addedto the residue. The mixture was fully stirred. The suspension was placedon a hemacytometer, and the number of cells per dish (surviving cellsand all cells) was counted twice. At that time, PBS and a trypan bluesolution were added, so that the number of cells contained in acompartment (1 mm×1 mm) to be observed could become approximately 100.The number of cells on two dishes was counted per day, and the meanvalue was obtained. Such measurement was carried out every 24 hours for4 days. The amounts of PBS and trypan blue suspended were changed tofacilitate observation of the cells every day. The depth of acompartment was 0.1 mm. Thus, the number of cells in 1 ml was obtainedby multiplying the volume of such a compartment (0.1 mm³) by 10⁴, andthe value obtained by multiplying the aforementioned number of cells bythe amount of a PBS-trypan blue solution/1000 (ml) was defined as thenumber of actually suspended cells. After completion of the observation,the obtained values were plotted on a semilogarithmic graph, so as toproduce a growth curve. Likewise, COS cells were dispersed on a 3.5-cmdish, resulting in a cell number of 1:10 (1.6×10⁵ cells), and they werethen observed for 4 days. From the logarithmic growth phase in thegrowth curve of each type of cells, a doubling time was obtained.

The results are shown in FIG. 3. As a result, the doubling times of FKDin a serum free medium, FKD in a serum medium, and COS in a serum mediumwere 19.0, 20.7, and 21.8 hours, respectively.

Example 6 Transfection of Mouse Interferon α and Purification ofExpressed Protein

FKD cells were cultured on a dish with a diameter of 10 cm(collagen-coated dish, IWAKI) up to a subconfluent state (approximately90% or more). The medium was removed, and 12 ml of DMEM-F12(+Supplement; penicillin/streptomycin were excluded) was then added tothe cells, followed by incubation. At the same time, 12 μg ofpHGGS-Mu-IFNα1 acting also as an expression plasmid of mouse interferon(hereinafter abbreviated as IFN), and a mixture obtained by adding 1.5ml of OPTI-MEM to the pHGGS-Mu-IFNα1, were produced. In addition, amixture obtained by mixing 36 μl of Lipofectamine 2000 (Invitrogen) and1.5 ml of OPTI-MEM was also produced. After leaving at rest for 5minutes, a Lipofectamine 2000 solution was added to the DNA solution,and the mixture was then left at rest for 20 minutes. Twenty minuteslater, the reaction solution was added to the incubated cells, and theobtained mixture was then stirred. Eight hours later, the medium wasexchanged with 10 ml of DMEM-F12 (+Supplement), and it was thenincubated for 16 hours.

Twenty-four hours after the transfection, the operation to recover themedium was repeated, and 200 ml of the medium was recovered as a sampleof IFN purification. The recovered medium was passed through a CPGcolumn (column size: 10 ml) obtained by filling a 10-ml syringe with CPG(Controlled-Pore Glass), so that the medium was adsorbed on the column.At that time, attention was paid that the flow rate of the medium pouredinto the column was kept at 6 to 10 ml. After 200 ml of the medium waspassed through the column, the inside of the column was washed with 100ml of 1×PBS. Thereafter, in order to elute IFN, 25 ml of 0.4 Mglycine-HCl (pH 3.5) was first poured therein, and 25 ml of 0.4 Mglycine-HCl (pH 2.0) was then poured therein. Mouse IFNα is eluted witha solution of pH 2.0, and IFNβ is eluted with a solution of pH 3.5. Inthe present experiment, since IFNα was synthesized in FKD cells, IFNαwas eluted to an eluted fraction of pH 2.0. In addition, since IFN hasthe property of adsorbing on a glass, a plastic container was used as acontainer for such an eluted fraction.

After completion of the elution, 25 ml of each eluted fraction wasdialyzed with 1×PBS. At that time, 1×PBS was exchanged twice. Aftercompletion of the dialysis, the resultant fraction was concentrated byAQUACIDE II (CALBIOCHEM), and it was then dialyzed with 1×PBS again.Thereafter, the fraction was centrifuged by Ultra-4 centrifugal filterunit (Amicon) at 5,000 rpm for 30 minutes, and thus each eluted fractionwas finally concentrated to 100 μl.

5 μL of a 5×SDS-PAGE sampling buffer (reductive condition) was added to20 μl of each eluted fraction, and the obtained mixture was then boiledfor 5 minutes to create a sample. Thereafter, SDS-PAGE was carried out.At that time, 25 μl of the sample was supplied to each lane of 15% gel,and the total amount of sample was supplied at 15 mA. Theelectrophoresed gel, six filters previously impregnated with a blottingbuffer, and a PVDF filter were laminated in the order of the threefilters, the PVDF filter, the gel, and the three filters from thebottom. Thereafter, transfer was carried out at a constant current of1.5 mA/cm² for 1 hour 30 minutes. After completion of the transfer, theresultant was immersed in a blocking buffer, so that a blockingtreatment was carried out at 4° C. overnight. After completion of theblocking treatment, an anti-mouse IFNα polyclonal antibody (rabbit) wasdiluted 500 times, and it was used as a primary antibody. The filter wasreacted with the antibody in a plastic pack at room temperature for 1hour. Thereafter, the filter was washed with TBS-T (5 minutes×5).Protein A-HRP (2000 times diluted) was used as a secondary antibody. Thefilter was reacted with the secondary antibody at room temperature for 1hour. After the filter was washed with TBS-T (5 minutes×5), the filterwas reacted with ECL plus (Amersham Biosciences), and was then exposedto an X-ray film. As a result of the analysis by Western blot, a band(approximately 20 kDa) was detected at a position of IFNα to which asugar chain was added. Thus, it was found that sugar chain-added IFNαwas secreted.

Example 7 Measurement of Biological Activity of IFN (CPE (CytopathicEffect) Assay; FIG. 4)

An IFN expression vector was transfected to FKD cells, and the thustransfected medium was added to L cells (Flow laboratories) so as tocause viral infection of the cells. After that, if the cells did notcause CPE due to viral infection, it can be said that active IFN existedin the medium, namely, activated IFN was secreted outside the FKD cells.

In order to conduct a CPE assay, diluted VSV was added to L cells, andtwenty-four hours later, the cells were observed. As a result, it wasconfirmed that CPE sufficiently occurs by 100 to 200 times dilution. AnIFN expression vector was transfected to FKD cells. The medium wasexchanged with a fresh one for 5 days, and the cells were thenrecovered. Thereafter, the cells were centrifuged at 3,000 rpm for 5minutes, and a supernatant was then recovered and used as a sample.

L cells were dispersed on a 96-well plate, resulting in a ratio of 1:2.After the cells had proliferated, 50 μl of IFN was added to theuppermost well in the longitudinal lane, and it was then stirred 10times. Thereafter, 50 μl of the reaction solution was transferred to alower well. This operation was continued until the solution reached thelowermost well, so as to produce a lane in which IFN was diluted 3 timesin a stepwise manner. The same above operation was carried out also on asample obtained after completion of the aforementioned transfection, sothat individual lanes were produced.

Six hours later, the medium was removed from each well, and 100 μl ofVSV that was 200 times diluted with 0.5% NCS-MEM was added to each well.At the same time, a control well of VSV was also prepared. Aftercompletion of the culturing for 24 hours, if it was confirmed that Lcells in the VSV control well had caused CPE, the cells were stainedwith crystal violet dye and were then dried. Thereafter, the ratio ofsurviving cells stained into violet was observed. A spot wherein 50% oftotal cells had been degenerated was identified from the wells of eachlane, and the approximate amount of IFN released (unit/ml) was assumed.In the case of COS cells, since it had been confirmed that IFN isreleased to outside the cells, the same operation was carried out alsoon such COS cells. A comparison was made between the two types of cells.The results are shown in FIG. 4. The 50% CPE spot of FKD cells wasalmost the same as that of COS cells. Thus, it was considered that thesecretion quantities of FKD cells were equivalent to those of COS cells(10,000 U/ml).

Example 8 Transfection of Cauxin and Expression of Protein

Using pcDNA6.2/GW/D-TOPO (Invitrogen), a Cauxin expression vector wasproduced. By using the cDNA of Cauxin as a template (it is to be notedthat the nucleotide sequence of Cauxin gene has been registered at theNCBI under Accession No. AB045377.), a full-length open reading framewas amplified by PCR (KOD-Plus from TOYOBO was used in PCR) using thefollowing primers:

(SEQ ID NO: 1) F-Primer: CACCATGAGTGGGATGTGGGTGCA; and (SEQ ID NO: 2)RV-Primer: TCAGGGGACAATGGTATTCA, andthe amplified product was then incorporated as an insert into the 950 nposition of the vector.

For transfection, the lipofection method was applied. FKD cells werecultured on a 3-cm dish up to a subconfluent state (approximately 80% to90%), and the medium was then removed. 1.5 ml of 5% FBS-DMEM-F12(-penicillin/streptomycin) was added, followed by incubation. At thesame time, 2 μl of CMV-cauxin (1 μg/μl) and a mixture obtained by adding250 μl of OPTI-MEM to the CMV-cauxin were produced. In addition, amixture obtained by mixing 6 μl of Lipofectamine 2000 (Invitrogen) and250 μl of OPTI-MEM was also produced. After leaving at rest for 5minutes, a Lipofectamine 2000 solution was added to the DNA solution,and the mixture was then left at rest for 20 minutes. Twenty minuteslater, the reaction solution was added to the incubated cells, and theobtained mixture was then stirred. Twenty hours later, the medium wasexchanged with DMEM-F12 and 5% FBS-DMEM-F12, and it was then incubatedfor 8 hours. Eight hours later, the medium (1.5 ml) was recovered, and 1ml of a soluble buffer was added to each dish. Thereafter, a sonictreatment was carried out for 5 minutes, and the reaction product wasthen centrifuged at 15,000 rpm for 10 minutes. Thereafter, a supernatantwas recovered, and it was used as a cell lysate (900 μl). With regard tothe medium, the medium was treated at 3,000 rpm for 5 minutes, so as toproduce a medium, from which dead cells had been eliminated.

Example 9 Treatment with Glycopeptidase F (GPF) (FIG. 5)

After immunoprecipitation with a cauxin C-terminal peptide antibody, 20μl of MQ water was added to a sample to be subjected to a glycosidasetreatment, and the obtained mixture was then stirred. Thereafter, 10 μlof the mixed solution was transferred into another tube. 10 μl of adenature buffer (+2-ME) was added thereto, and the mixture was thenboiled at 100° C. for 3 minutes. Thereafter, 20 μl of a stabilizedsolution and 52 μl of MQ water were added thereto, and the mixture wasstirred. Thereafter, 8 μl (0.5 mU/μl) of GPF (TAKARA) was added to themixture, and the obtained mixture was then stirred, followed byincubation at 37° C. for 20 hours. Thereafter, 25 μl of 5×SDS-PAGEsampling buffer (reductive condition) was added, and the obtainedmixture was then boiled for 5 minutes to create a sample. Thereafter, asupernatant was recovered, and SDS-PAGE was carried out. At that time,10 μl of the sample was supplied to each lane of 10% gel, and the totalamount of sample was supplied at 15 mA. After completion of theelectrophoresis, three filters, a membrane, a gel, and three filterswere laminated in this order. Transfer was carried out at a constantcurrent of 1.5 mA/cm² for 1 hour 30 minutes. After completion of thetransfer, the resultant was immersed in a blocking buffer, so that ablocking treatment was carried out at 4° C. overnight.

After completion of the blocking treatment, a cauxin C-terminal peptideantibody (8,000 times diluted) was used as a primary antibody. Thefilter was reacted with the antibody in a plastic pack at roomtemperature for 1 hour. Thereafter, the filter was washed with TBS-T (5minutes×5). Protein A-HRP (8,000 times diluted) was used as a secondaryantibody. The same treatment as in the case of the primary antibody wascarried out. After the filter was washed with TBS-T (5 minutes×5), thefilter was reacted with ECL purchased from Amersham Biosciences) and wasexposed to an X-ray film. The results are shown in FIG. 5. From theresults as shown in FIG. 5, it was confirmed that a sugar chain wasadded to a cauxin protein allowed to express in the FKD cells of thepresent invention.

Example 10 Suspension Culturing of FKD Cells

FKD cells were cultured on four 10-cm dishes up to an almost confluentstate (medium: DMEM F-12 (10565-018, GIBCO; 5% FCS). The medium wasremoved, and the cells were washed with 5 ml of PBS-EDTA. Thereafter, 1ml of 0.1% trypsin/PBS-EDTA was added thereto, and the mixture was thenincubated in a CO₂ incubator for 4 minutes. The cells released from thedishes were recovered using a P1000 PIPETMAN, and they were then placedin a 50-ml centrifuge tube that contained 10 ml of a serum free medium(OPTI PRO SFM, 12309-019, GIBCO) comprising 5 mg of a soybean trypsininhibitor (Wako 202-09221). The cells were then centrifuged at 100 g for4 minutes. Thereafter, a supernatant was removed, and the cells weresuspended in 10 ml of a serum free medium (as described above), and thesuspension was then centrifuged at 100 g for 4 minutes. Thereafter, asupernatant was removed, and the cells were then resuspended in 10 ml ofa serum free medium. The suspension was added to a spinner flask(S-flask 4500-250, TAITEC) that contained 90 ml of a serum free mediumcomprising 1 ml of Pluronic F-68 (11905-031, GIBCO), and the cells werethen cultured in a CO₂ incubator (5% CO₂, 37° C.) at a stirrer rotationnumber of 90 rpm. The state of the cells from day 1 to day 4 afterinitiation of the suspension culturing is shown in FIG. 6.

The number of cells was measured as follows. 1 ml of a culture solutionwas centrifuged at 100 g×4 minutes, so as to recover cells. Therecovered cells were washed with 1 ml of PBS-EDTA, and 1 ml of 0.1%trypsin/PBS-EDTA was added thereto. Thereafter, the obtained mixture wasleft at room temperature for 5 minutes, so that cell masses weredispersed. Thereafter, the number of cells was measured using ahemacytometer. The results obtained by measuring the number of cellsfrom day 1 to day 5 after initiation of the suspension culturing areshown in FIG. 7. As shown in FIG. 7, cell growth was observed until day4 to day 5 after initiation of the suspension culturing.

INDUSTRIAL APPLICABILITY

The established cell line of the present invention is able toproliferate in a serum free medium, is able to synthesize and secrete alarge amount of protein of interest, and is able to produce aglycoprotein having a sugar chain structure. Accordingly, theestablished cell line of the present invention is useful as a host cellused for production of a recombinant protein. In particular, using theestablished cell line of the present invention as a host cell, itbecomes possible to produce a large amount of protein of interest at alow cost in the form of the protein having biological activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows FKD cells.

FIG. 2 shows the results obtained by analyzing the gene transductionefficiency of FKD cells, COS cells, and HeLa cells. To each type ofcells, lacZ was introduced, and the cells were then stained with X-gal.Cell density was the same in all of the three types of cells.

FIG. 3 shows the results obtained by comparing the growth of FKD cellswith the growth of COS cells. The rhombus indicates FKD cells (serumfree), the square indicates FKD cells (serum), and the triangleindicates COS cells (serum).

FIG. 4 shows the results obtained by analyzing the expression of MuIFN-α1 in FKD cells and in COS cells.

FIG. 5 shows the results obtained by analyzing the expression ofglycoprotein Cauxin. Using an anti-Cauxin antibody, Western blotting wascarried out regarding a case where Endo F (sugar chain cleavage enzyme)was added to a FKD cell extract, in which Cauxin gene had beenexpressed, and a case where no Endo F was added to such a FKD cellextract. Endo F (Endoglycosidase F) specifically cleaves the bindingsite (GlcNAc-Asn bond) of an N-glycoside sugar chain with a protein.This enzyme cleaves such a binding site regardless of the structure of asugar chain portion, except for a sugar chain in which fucose binds toGluNAc at a reducing terminal via α1-3 bond.

FIG. 6 shows the state of FKD cells on day 1 (a), day 2 (b), day 3 (c),and day 4 (d) after initiation of the suspension culturing of the cells.

FIG. 7 shows the results obtained by measuring the number of FKD cellsfrom day 1 to day 5 after initiation of the suspension culturing of thecells.

1. An established cell line, which is established from distal tubularepithelial cells derived from a mammal.
 2. The established cell lineaccording to claim 1, which is used as a host cell for production of arecombinant protein.
 3. The established cell line according to claim 1,which is derived from a cat.
 4. The established cell line according toclaim 1, which has a gene encoding a foreign immortality-impartingfactor.
 5. The established cell line according to claim 1, which has aforeign selective marker gene.
 6. The established cell line according toclaim 1, which is able to proliferate in a serum free medium.
 7. Theestablished cell line according to claim 1, which is a cell having anaccession No. FERM BP-10643.
 8. A transformant, which is obtained byintroducing a gene encoding a protein of interest to be expressed intothe established cell line of claim
 1. 9. A method for producing arecombinant protein using the established cell line of claim
 1. 10. Themethod according to claim 9, which comprises: introducing a foreign geneinto a cell line established from distal tubular epithelial cellsderived from a mammal to produce transformed cells; culturing theobtained transformed cells, so as to allow the cells to produce arecombinant protein encoded by the foreign gene; and recovering theproduced recombinant protein.
 11. The method according to claim 9,wherein the produced recombinant protein is modified with a sugar chain.12. A recombinant protein, which is produced by the method of claim 9.